Nuclear factor of κB (NF-κB) is a family of ubiquitously expressed transcription factors that are rapidly activated in response to several biological stimuli including inflammatory cytokines, bacterial and viral infections and other extracellular signals. NF-κB and related family members are involved in the regulation of more than 50 genes relating to immune and inflammatory responses ((Barnes P J, Karin M (1997) N Engl J Med 336, 1066-1071) and (Baeuerle P A, Baichwal V R (1997) Adv Immunol 65, 111-137)). Activation of NF-κB is regulated by an inhibitor of κB kinase (IKK) complex. Pro-inflammatory signals activate the IKK complex via a cascade of protein phosphorylations that result in increased catalytic activity; the complex in turn phosphorylates the NF-κB-bound IκB. Phosphorylation of IκB facilitates its ubiquitination and subsequent degradation by the proteasome. Freed from IκB, the active NF-κB is able to translocate to the nucleus where it binds in a selective manner to preferred gene-specific enhancer sequences and drives the transcription of a number of genes (Reviewed by Ghosh and Karin in Cell (2002) 109: S81-S96).
Phosphorylation of IκB by the IKK complex that influences cytoplasmic to nuclear translocation of NF-κB is therefore a key regulatory step in the signal transduction pathway. The IKK complex consists of two IκB kinases, IKKα (IKK1), IKKβ (IKK2), and a scaffolding protein, IKKγ (NEMO) which has no known catalytic activity. IKKα and IKKβ phosphorylate IκBs on specific serine residues to initiate protein degradation. On IκBα, phosphorylation occurs on two serine residues: Ser32 and Ser36. Studies with IκBα mutants that cannot be phosphorylated on these serine residues show they block NF-κB activation by acting as dominant-negative derivatives. Mutants of IKKα and IKKβ that act as dominant-negative derivatives also block the activation of NF-κB in cells. Thus, inhibitors of IκB kinases that prevent IκB phosphorylation would similarly block NF-κB activation and a number of such inhibitors have now been described (recently reviewed by Karin, Yamamoto and Wang in Nature Reviews (2004) 3: 17-26). Such inhibitors would be useful for treating inflammatory disorders mediated through NF-κB-dependent gene transcription.
Among the genes driven by NF-κB are several that encode for proteins that are implicated in inflammation such as cytokines TNFα, IL-1β, IL-6, IL-8; adhesion molecules such as ICAM-1, v-CAM-1, E-selectin; and enzymes such as iNOS, cPLA2 and Cox-2 (Reviewed by Pahl in Oncogene (1999) 49: 6853-6866). Normally, the inflammatory process is a localized response to tissue injury or infection that leads to recruitment of blood cells and accumulation of fluid at the site of injury that ultimately results in healing. In certain instances, however, over-activity or dysfunction of the normal inflammatory response leads to exacerbation and causes harm that results in diseased states. NF-κB has been shown to be activated in a number of inflammatory diseases. As NF-κB drives the expression of a number of key molecules implicated in inflammation and immune response, inhibition of its activation under such diseased states would block the underlying inflammation and prevent, halt or reverse the disease. This broad anti-inflammatory activity of NF-κB would be advantageous over current treatment options such as NSAIDs that treat the symptoms but not the underlying causes of the disease. NF-κB has been reported to be a key link between inflammation and cancer (reviewed by Li et al., in Trends in Immunology (2005) 26, 318-325; Greten and Karin (2004) 206, 193-199). NF-κB drives several genes that promote cell survival such as c-IAP-1, c-IAP-2, Bcl-XL and p53 and a number of genes that promote proliferation such as cyclin-D1 and c-myc. The transcription factor has been reported to be constitutively activated in a number of cancers including breast, prostate and melanoma. Activation of other pathways that have been implicated in cancer such as HER2, IGF-1, Ras and Akt has also been reported to result in NF-κB activation. Furthermore, anti-neoplastic agents have been demonstrated to result in the activation of NF-κB. Thus, inhibiting NF-κB would have significant advantages over current treatment options in cancer therapy as a chemopreventive, chemosensitizer, and a therapeutic agent in cancers including cancer of the breast, prostate and skin.
The JANUS (JAKs) family of proteins are comprised of 7 homology domains including 2 kinase domains; a catalytic (JH1) and a pseudo kinase domain (JH2) that is devoid of catalytic activity. Currently, there are four known mammalian JAK family members: JAK1, JAK2, JAK3 and TYK2. JAK1, JAK2 and TYK2 are ubiquitously expressed whereas JAK3 is expressed in the myeloid and lymphoid lineages. The JAK family members are non-receptor tyrosine kinases that associate with many cell surface receptors such as hematopoietin cytokines, receptor tyrosine kinases and GPCR's (see Table 1) which regulate diverse cell processes including migration, proliferation, differentiation, and survival. Binding of the ligand to their respective extracellular receptor leads to the recruitment of a JAK protein and subsequent phosphorylation of both the receptor and the JAK protein. The STATs (known as signal transducers and activators of transcription protein), which are the main downstream effectors of JAK, are recruited by pJAK leading to the phosphorylation and dimerization of the STAT proteins which subsequently translocate to the nucleus and drive gene transcription.
TABLE 1LigandsJAK KinasesStatsIFN familyIFN-α,β,γ,limitinTYK2, JAK1STAT1, STAT2 (STAT3,STAT 4, STAT 5)IFN-χJAK1, JAK2STAT1 (STAT5)IL-10TYK2, JAK1STAT3IL-19undefinedundefinedIL-20undefinedSTAT3IL-22undefinedSTAT3, STAT5Gp130 familyIL-6JAK1, JAK2STAT3, STAT1IL-11JAK1STAT3, STAT1OSMJAK1, JAK2STAT3, STAT1LIFJAK1, JAK2STAT3, STAT1CNTFJAK1, JAK2STAT3, STAT1NNT-1/BSF-3JAK1, JAK2STAT3, STAT1G-CSFJAK1, JAK2STAT3CT-1JAK1, JAK2STAT3LeptinJAK2STAT4IL-12TYK2, JAK2STAT4IL-23undefinedSTAT4χC familyIL-2JAK1, JAK3STAT5, STAT3IL-7JAK1, JAK3STAT5, STAT3TSLPundefinedSTAT5IL-9JAK1, JAK3STAT5, STAT3IL-15JAK1, JAK3STAT5, STAT3IL-21JAK1, JAK3STAT5, STAT3, STAT1IL-4JAK1, JAK3STAT6IL-13JAK1STAT6, STAT3IL-3 familyIL-3JAK2STAT5IL-5JAK2STAT5GM-CSFJAK2STAT5Single chain familyEPOJAK2STAT5GHJAK2STAT5, STAT3PRLJAK2STAT5TPOJAK2STAT5Receptor tyrosine kinasesEGFJAK1, JAK2STAT1, STAT3, STAT5PDGFJAK1, JAK2STAT1, STAT3CSF-1TYK2, JAK1STAT1, STAT3, STAT5HGFundefinedG-protein coupled receptorsAT1JAK2STAT1, STAT2
JAK1−/− mice were found to be developmentally similar to the JAK1+/+ although they weighed 40% less than the wild-type and failed to nurse at birth. These pups were not viable and died within 24 hours of birth (Meraz et al Cell, 1998, 373-383). JAK1 deficiency led to reduced number of thymocytes, pre-B cells and mature T and B lymphocytes. TYK2(−/−) mice, on the other hand, are viable, demonstrating subtle defects in their response to IFN-α/β and IL-10 and profound defects to the response of IL-12 and LPS.
The breast cancer susceptibility protein (BRCA1) acts as a tumor suppressor and contributes to cell proliferation, cycle regulation, as well as DNA damage and repair. BRCA1 (−/−) mice develop normally but die by 7.5 days post embryo suggesting a key role of BRCA1 for development. Mice in which the BRCA1 protein was overexpressed led to inhibition of cell growth and sensitized cells to cytotoxic reagents. In the human proSTATe cancer cell line Du-145 (Gao FEBS Letters 2001, 488, 179-184), enhanced expression of BRCA1 was found to correlate with constitutive activation of STAT3 as well as activation of JAK1 and JAK2. Moreover, antisense oligonucleotides selective for STAT3 led to significant inhibition of cell proliferation and apoptosis in Du-145 cells. This data supports the potential utility of JAK1 and JAK2 inhibitors in the treatment of proSTATe cancer.
Campbell et al (Journal of Biological Chemistry 1997, 272, 2591-2594) as reported that STAT3 is constitutively activated v-Src transformed cells. To test whether STAT3 activation resulted via signaling through the JAK-STAT pathway, three fibroblast cell lines (NIH3T3, Balb/c, and 3Y1) were transformed with v-Src. The level of JAK1 phosphorylation in NIH3T3 cells was markedly increased in cells overexpressed with v-Src or mutant c-Src (Y527F) compared to those in the less transforming c-Src. This result correlated with increased JAK1 enzymatic activity. Similar results were observed with JAK2 albeit to a lesser extent. These results are consistent with constitutive activation of JAK1 and possibly JAK2 which contribute to the hyperactivation of STAT3 in Src-transformed cells.
Asthma is a disease that is increasing in prevalence and results in “airway obstruction, airway hyperresponsiveness, and airway inflammation and remodeling” (Pernis The Journal of Clinical Investigation 2002, 109, 1279-1283). A common cause is the inappropriate immune responses to environmental antigens usually involving CD4+ T helper cells (TH2) which are triggered from cytokines IL-4, IL-5, IL-6, IL-10, and IL-13 which signal through JAK1/JAK3-STAT6 pathway. Th1 cells are thought to be involved with the “delayed-type hypersensitivity responses” which secrete IL-2, IFN-γ, and TNF-β and signal through the JAK2/TYK2-STAT4 pathway. STAT6 (−/−) mice were protected from AHR when challenged with environmental antigens and showed no increase in IgE levels or the quantity of mucous containing cells.
Studies have disclosed an association between an activating JAK2 mutation (JAK2V617F) and myleoproliferative disorders (Gilliland Cancer Cell 2005). The myeloproliferative disorders, a subgroup of myeloid malignancies, are clonal stem cell diseases characterized by an expansion of morphologically mature granulocyte, erythroid, megakaryocyte, or monocyte lineage cells. Myeloproliferative disorders (MPD) include polycythemia vera (PV), essential thrombocythemia (ET), myeloid metaplasia with myelofibrosis (MMM), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome (HES), juvenile myelomonocytic leukemia (JMML) and systemic mast cell disease (SMCD). It has been suggested that abnormalities in signal transduction mechanisms, including constitutive activation of protein tyrosine kinases, initiate MPD.
JAK3 associates with the common gamma chain of the extracellular receptors for the following interleukins: IL-2, IL4, IL-7, IL-9 and IL-15. A JAK3 deficiency is associated with an immune compromised (SCID) phenotype in both rodents and humans. The SCID phenotype of JAK3−/− mammals and the lymphoid cell specific expression of JAK3 are two favorable attributes of a target for an immune suppressant. Data suggests that inhibitors of JAK3 could impede T-cell activation and prevent rejection of grafts following transplant surgery, or to provide therapeutic benefit to patients suffering autoimmune disorders.